Apongopus extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a preparation method of an aspongopus extract. The invention provides a preparation method of an aspongopus extract, which comprises the following steps: mixing an aspongopus raw material with water, sealing, refrigerating, boiling, cooling, carrying out solid-liquid separation, carrying out primary centrifugation, taking supernate, carrying out heating treatment, carrying out secondary centrifugation, taking supernate, mixing with a protease inhibitor, and carrying out ultrafiltration treatment after mixing to obtain the aspongopus extract. The molecular weight of the aspongopus extract is larger than 10 kDa and smaller than 30 kDa. According to the preparation method, the utilization rate of the aspongopus raw material can be increased, the requirements for raw materials and equipment are low, dried aspongopus or aspongopus extraction residues can be used as raw materials, and the preparation method is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a Aspongopus chinensis Dallas extract, a preparation method and application thereof. BACKGROUND

[0002] Breast cancer is one of the common malignant tumors that endanger human health, and seriously endangers human health. The current treatment methods for breast cancer include surgery, chemotherapy, radiotherapy, etc. Although surgery is the preferred solution for treating breast cancer, for those who cannot tolerate surgery or those with advanced tumor metastasis, chemotherapy is the best choice. However, during the process of chemotherapy for breast cancer, the problem of drug resistance has to be faced. Therefore, the development of natural drugs to overcome drug resistance and improve the sensitivity of chemotherapy drugs has become a hot research topic in the field of anti-breast cancer.

[0003] Traditional Chinese medicine has relatively few side effects, and has the characteristics of multi-pathway and multi-target anti-tumor, which is expected to overcome tumor chemotherapy resistance. At present, studies have shown that traditional Chinese medicine Huangqi extract can improve the sensitivity of cisplatin-resistant gastric cancer cell SGC-7901 / DDP to cisplatin, so traditional Chinese medicine has broad application prospects in anti-tumor treatment.

[0004] Aspongopus chinensis Dallas is a traditional Chinese medicine recorded in Shennong's Herbal Classic, which has the characteristics of attacking hard and breaking accumulation, and has therapeutic effects on "deficiency" and "evil accumulation" of tumors. The existing technology shows that Aspongopus chinensis Dallas extract has therapeutic effects on various tumors, including lung cancer, liver cancer, gastric cancer, breast cancer, etc.; Aspongopus chinensis Dallas water extract can inhibit the proliferation, migration and invasion of non-small cell lung cancer cells, and can induce apoptosis of liver cancer cells and breast cancer cells, thereby playing an anti-tumor role.

[0005] However, Aspongopus chinensis Dallas mainly relies on artificial breeding, and has low yield and high price. Moreover, the current extraction method of Aspongopus chinensis Dallas cannot fully extract the active ingredients in Aspongopus chinensis Dallas, and there are still a large amount of active ingredients in the worm residues after extraction, which causes resource waste. Therefore, how to provide a method for further fully extracting the effective components in Aspongopus chinensis Dallas to improve the utilization rate of Aspongopus chinensis Dallas and reduce the preparation cost of Aspongopus chinensis Dallas extract has become a problem to be solved in the field. SUMMARY

[0006] In order to improve the extraction efficiency of Aspongopus chinensis Dallas extract, the present application provides an Aspongopus chinensis Dallas extract, a preparation method and application thereof, which specifically comprises the following technical solutions:

[0007] A preparation method of Aspongopus chinensis Dallas extract, comprising the following steps:

[0008] Mixing Aspongopus chinensis Dallas raw materials with water, sealing and refrigerating to obtain a first mixture;

[0009] boiling the first mixture, and separating the solid and the liquid after cooling to obtain the water extract of aspongopus;

[0010] centrifuging the water extract of aspongopus for the first time to obtain a first supernatant and a first precipitate, and collecting the first supernatant;

[0011] heating the first supernatant to obtain a heated solution;

[0012] centrifuging the heated solution for the second time to obtain a second supernatant and a second precipitate;

[0013] mixing the second supernatant and a protease inhibitor to obtain a second mixture;

[0014] ultrafiltrating the second mixture to collect components with molecular weight >10 kDa and <30 kDa to obtain the aspongopus extract.

[0015] Preferably, the aspongopus raw material comprises one or more of aspongopus dry insects and aspongopus residues;

[0016] The aspongopus residues comprise insect body residues after extracting aspongopus blood lymph;

[0017] The mass-volume ratio of the aspongopus raw material to water is 40-60 g: 100 mL.

[0018] Preferably, the temperature of the refrigeration treatment is 4-6℃, and the time of the refrigeration treatment is 20-28 h.

[0019] Preferably, the boiling time is 0.5-1.5 h.

[0020] Preferably, the rotation speed of the first centrifugation and the second centrifugation is 10,000-14,000 rpm respectively, the temperature of the first centrifugation and the second centrifugation is 3-5℃ respectively, and the time of the first centrifugation and the second centrifugation is 10-20 min respectively.

[0021] Preferably, the heating treatment comprises metal bath treatment, alcohol lamp heating treatment or water bath treatment.

[0022] When the heating treatment is metal bath treatment, the temperature of the metal bath treatment is 80-120℃, and the time of the metal bath treatment is 25-35 min.

[0023] Preferably, the volume ratio of the protease inhibitor to the second supernatant is 1-5: 100-500.

[0024] Preferably, the ultrafiltration treatment comprises: performing first ultrafiltration treatment on the second mixture by using a 30kDa ultrafiltration device to obtain a first component with a molecular weight of 30kDa or above and a second component with a molecular weight of less than 30kDa;

[0025] performing second ultrafiltration treatment on the second component with a molecular weight of less than 30kDa by using a 10kDa ultrafiltration device to obtain a third component with a molecular weight of less than 10kDa and a fourth component with a molecular weight of more than 10kDa and less than 30kDa, and collecting the fourth component to obtain the extract of asiaspidia spp.

[0026] Preferably, the first ultrafiltration treatment is performed for 20 min, the rotation speed of the first ultrafiltration treatment is 35000 rpm, the temperature of the first ultrafiltration treatment is 4 DEG C, and the 30kDa ultrafiltration device comprises a 30kDa double-vertical filtration membrane ultrafiltration tube.

[0027] The second ultrafiltration treatment is performed for 20 min, the rotation speed of the second ultrafiltration treatment is 5000 rpm, the temperature of the second ultrafiltration treatment is 4 DEG C, and the 10kDa ultrafiltration device comprises a 10kDa double-vertical filtration membrane ultrafiltration tube.

[0028] The application further provides application of the extract of asiaspidia spp. prepared by the preparation method to preparation of an antitumor drug.

[0029] The application has the following advantages:

[0030] The application provides a preparation method of an extract of asiaspidia spp., which comprises the following steps: mixing asiaspidia spp. raw materials with water, sealing, performing cold storage treatment, and obtaining a first mixture; boiling the first mixture, performing solid-liquid separation after cooling, and obtaining asiaspidia spp. water extract; performing first centrifugation on the asiaspidia spp. water extract, and obtaining first supernatant and first precipitate; performing metal bath treatment on the first supernatant, and obtaining a heated solution; performing second centrifugation on the heated solution, and obtaining second supernatant and second precipitate; mixing the second supernatant with a protease inhibitor, and obtaining a second mixture; performing ultrafiltration treatment on the second mixture, and obtaining an extract of asiaspidia spp. with a molecular weight of more than 10kDa and less than 30kDa. The preparation method can improve the utilization rate of asiaspidia spp. raw materials; the preparation method can complete the extraction of the extract of asiaspidia spp. within 2h, greatly shortening the preparation period of the extract of asiaspidia spp.; the preparation method has low requirements on processing instruments, only needs a centrifuge and a water bath kettle, has low preparation cost, is suitable for industrial production, and provides a new idea for asiaspidia spp. processing. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows.

[0032] Figure 1 SDS-PAGE electrophoresis result of the aspongopus extract in the embodiment 2 of the present application; wherein, M represents Marker, and CHP represents the aspongopus extract;

[0033] Figure 2 Influence of different groups on the proliferation of breast cancer cells in the embodiment 3 of the present application, ** represents P<0.01, indicating that the difference is extremely significant compared with the control group;

[0034] Figure 3 Proliferation inhibition result of different ferroptosis inhibitors on MCF-7 by the aspongopus extract in the embodiment 4 of the present application; wherein, * represents P<0.05, indicating that the difference is significant compared with the control group; ** represents P<0.01, indicating that the difference is extremely significant compared with the control group; # represents P<0.05, indicating that the difference is significant compared with the aspongopus extract group; ## represents P<0.01, indicating that the difference is extremely significant compared with the aspongopus extract group.

[0035] Figure 4 Detection result (200x) of ROS of breast cancer cells by using DCFH-DA fluorescent probe in the embodiment 5 of the present application; wherein, (A) is that the aspongopus extract improves the ROS level in breast cancer SUM-159 cells; (B) is that the aspongopus extract improves the ROS level in breast cancer MDA-MB-453 cells; (C) is that the aspongopus extract improves the ROS level in breast cancer MCF-7 cells; (D) is that the aspongopus extract improves the ROS level in breast cancer MCF-7 / ADR cells;

[0036] Figure 5 Fe2+ detection result (200x) of breast cancer cells by using RhoNox-1 fluorescent probe in the embodiment 5 of the present application; wherein, (A) is that the aspongopus extract improves the Fe2+ content in breast cancer SUM-159 cells; (B) is that the aspongopus extract improves the Fe2+ content in breast cancer MDA-MB-453 cells; (C) is that the aspongopus extract improves the Fe2+ content in breast cancer MCF-7 cells; (D) is that the aspongopus extract improves the Fe2+ content in breast cancer MCF-7 / ADR cells. 2+ 2+ 2+ 2+ 2+ DETAILED DESCRIPTION

[0037] The present application provides a preparation method of aspongopus extract, comprising the following steps: ​​​​​

[0038] Mixing the aspongopus raw material with water and sealing, refrigeration, to obtain the first mixture; boiling the first mixture, solid-liquid separation after cooling, to obtain the aspongopus water extract; the first supernatant and the first precipitate are obtained by centrifuging the aspongopus water extract for the first time, and the first supernatant is collected; the first supernatant is heated to obtain a heated solution; the second supernatant and the second precipitate are obtained by centrifuging the heated solution for the second time; the second supernatant is mixed with protease inhibitors to obtain a second mixture; the components with molecular weight > 10 kDa and < 30 kDa are collected by ultrafiltration treatment of the second mixture to obtain the aspongopus extract.

[0039] Mixing the aspongopus raw material with water and sealing, refrigeration, to obtain the first mixture; boiling the first mixture, solid-liquid separation after cooling, to obtain the aspongopus water extract; the first supernatant and the first precipitate are obtained by centrifuging the aspongopus water extract for the first time; the heated solution is obtained by metal bath treatment of the first supernatant; the second supernatant and the second precipitate are obtained by centrifuging the heated solution for the second time; the second mixture is obtained by mixing the second supernatant with protease inhibitors; the components with molecular weight in the range of 10 kDa to 30 kDa are collected by ultrafiltration treatment of the second mixture to obtain the aspongopus extract.

[0040] The present application mixes the asion raw material with water and seals it, and then performs refrigeration treatment to obtain a first mixture. As an optional embodiment, the asion raw material includes one or more of asion dried insects and asion residues. As an embodiment, the asion residues include insect body residues after extracting asion blood lymph. As an embodiment, the insect body residues after extracting asion blood lymph are: the head and wings of the frozen asion are separated, the insect body is used to extract blood lymph, and the remaining residue part is used as an extraction raw material. In the case of a shortage of fresh asion, the use of residues can alleviate the shortage of asion. As a preferred embodiment, the asion raw material is the insect body residues after extracting asion blood lymph. As an embodiment, the mass-volume ratio of the asion raw material to water is 40-60g:100mL. As an optional embodiment, the mass-volume ratio of the asion raw material to water can be 40g:100mL, 41g:100mL, 42g:100mL, 43g:100mL, 44g:100mL, 45g:100mL, 46g:100mL, 47g:100mL, 48g:100mL, 49g:100mL, 50g:100mL, 51g:100mL, 52g:100mL, 53g:100mL, 54g:100mL, 55g:100mL, 56g:100mL, 57g:100mL, 58g:100mL, 59g:100mL or 60g:100mL. As an embodiment, the sealing can be sealed with a preservative film. As an embodiment, the refrigeration treatment temperature is 4-6℃. As an optional embodiment, the refrigeration treatment temperature can be 4℃, 5℃ or 6℃. As an embodiment, the refrigeration treatment time is 20-28h. As an optional embodiment, the refrigeration treatment time can be 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h or 28h. In a specific embodiment, the present application mixes asion and water and soaks it at 4℃ for 24h, so that the water-soluble substances in asion are fully dissolved in water.

[0041] After obtaining the first mixture, the present application boils the first mixture, and after cooling, solid-liquid separation is performed to obtain the water extract of asion. As an embodiment, the boiling time is 0.5-1.5h. As an alternative embodiment, the boiling time can be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h. As an embodiment, the boiling includes placing the first mixture in a beaker, placing the beaker on a tripod with asbestos net, and heating with an alcohol lamp to boil for 1h. As an embodiment, the cooling is cooling to room temperature. As an embodiment, the method of solid-liquid separation includes filtration, and the filtrate is collected. As an embodiment, the operation of filtration includes guiding the liquid in the beaker into a centrifuge tube with a glass funnel and filter paper.

[0042] After obtaining the water extract of asion, the present application performs first centrifugation on the water extract of asion to obtain first supernatant and first precipitate, and the first supernatant is collected. As an embodiment, the rotation speed of the first centrifugation is 10000-14000rpm. As an alternative embodiment, the rotation speed of the first centrifugation can be 10000rpm, 11000rpm, 12000rpm, 13000rpm or 14000rpm. As an embodiment, the temperature of the first centrifugation is 3-5℃. As an alternative embodiment, the temperature of the first centrifugation can be 3℃, 4℃ or 5℃. As an embodiment, the time of the first centrifugation is 10-20min. As an alternative embodiment, the time of the first centrifugation can be 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min. As an embodiment, after collecting the first supernatant, the first precipitate is further stored in an environment of-80℃, and used for other experimental steps.

[0043] After obtaining the first supernatant, the present application heats the first supernatant to obtain a heated solution. As an embodiment, the heating method can be metal bath, water bath or alcohol lamp heating. As an embodiment, when the heating method is metal bath, the temperature of the metal bath treatment is 80-120°C. As an alternative embodiment, the temperature of the metal bath treatment can be 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C or 120°C. As an embodiment, the time of the metal bath treatment is 25-35 min. As an alternative embodiment, the time of the metal bath treatment can be 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min or 35 min.

[0044] After obtaining the heated solution, the present application centrifuges the heated solution for the second time to obtain a second supernatant and a second precipitate. As an embodiment, the speed of the second centrifugation is 10000-14000 rpm. As an alternative embodiment, the speed of the second centrifugation can be 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm. As an embodiment, the temperature of the second centrifugation is 3-5°C. As an alternative embodiment, the temperature of the second centrifugation can be 3°C, 4°C or 5°C. As an embodiment, the time of the second centrifugation is 10-20 min. As an alternative embodiment, the time of the second centrifugation can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min. As an embodiment, after the second centrifugation, the present application further comprises the steps of collecting the second supernatant and discarding the second precipitate.

[0045] After obtaining the second supernatant, the present application mixes the second supernatant and a protease inhibitor to obtain a second mixture. As an embodiment, the volume ratio of the protease inhibitor to the second supernatant is 1-5:100-500. As an alternative embodiment, the volume ratio of the protease inhibitor to the second supernatant can be 1:100, 1:200, 1:300, 1:400, 1:500, 2:100, 2:200, 2:300, 2:400, 2:500, 3:100, 3:200, 3:300, 3:400, 3:500, 4:100, 4:200, 4:300, 4:400, 4:500, 5:100, 5:200, 5:300, 5:400 or 5:500.

[0046] After obtaining the second mixture, the present application performs ultrafiltration treatment on the second mixture, and collects components with molecular weight >10 kDa and <30 kDa to obtain the asal extract. As an embodiment, the ultrafiltration treatment comprises: performing first ultrafiltration treatment on the second mixture by using a 30 kDa ultrafiltration device to obtain first components with molecular weight ≥30 kDa and second components with molecular weight <30 kDa; performing second ultrafiltration treatment on the second components with molecular weight <30 kDa by using a 10 kDa ultrafiltration device to obtain third components with molecular weight <10 kDa and fourth components with molecular weight >10 kDa and <30 kDa, and collecting the fourth components to obtain the asal extract. Through previous experiments, the applicant finds that the asal extract with molecular weight of 10-30 kDa has an anticancer effect, and thus the extract with molecular weight of 10-30 kDa is collected.

[0047] As an embodiment, the first ultrafiltration treatment has a time of 10-20 min. As an alternative embodiment, the first ultrafiltration treatment can have a time of 10 min, 11 min, 12 min, 13 min, 14 min or 15 min. As an embodiment, the first ultrafiltration treatment has a rotation speed of 3000-5000 rpm. As an alternative embodiment, the first ultrafiltration treatment can have a rotation speed of 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm or 5000 rpm. As an embodiment, the first ultrafiltration treatment has a temperature of 3-5 ℃; as an alternative embodiment, the first ultrafiltration treatment can have a temperature of 3 ℃, 4 ℃ or 5 ℃. As an embodiment, the 30 kDa ultrafiltration device comprises a 30 kDa double vertical filtration membrane ultrafiltration tube. As an embodiment, the second ultrafiltration treatment has a time of 10-20 min. As an alternative embodiment, the second ultrafiltration treatment can have a time of 10 min, 11 min, 12 min, 13 min, 14 min or 15 min. As an embodiment, the second ultrafiltration treatment has a rotation speed of 3000-5000 rpm. As an alternative embodiment, the second ultrafiltration treatment can have a rotation speed of 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm or 5000 rpm. As an embodiment, the second ultrafiltration treatment has a temperature of 3-5 ℃; as an alternative embodiment, the second ultrafiltration treatment can have a temperature of 3 ℃, 4 ℃ or 5 ℃.

[0048] The present application also provides use of the asal extract prepared by the preparation method as described above in the preparation of an antitumor drug.

[0049] In order to further illustrate the present application, a preparation method of a blister beetle extract and applications thereof provided by the present application are described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0050] Example 1: A preparation method of a blister beetle extract

[0051] After the blood lymph of the blister beetle is extracted, the insect body residues and deionized water are mixed at a mass-volume ratio of 50g:100mL, and then added into a beaker. The beaker is soaked at 4℃ for 24h to make the water-soluble substances in the blister beetle fully dissolved in water. The beaker is placed on a tripod with an asbestos net, and heated with an alcohol lamp to boil for 1h. After cooling, the insect body residue mixture is obtained.

[0052] The insect body residue mixture is introduced into a 50ml centrifuge tube, and centrifuged at 12000rpm and 4℃ for 15min to obtain a first supernatant and a first precipitate. The above steps are repeated for 3 times until no precipitate is obtained.

[0053] Protease inhibitors (Bi Yun Tian, P1005) are added into the centrifuge tube at a volume ratio of 1:100 with the second supernatant. The supernatant containing the protease inhibitors is transferred into a 30kDa double vertical filtration membrane ultrafiltration tube using a 1ml pipette. The 30kDa double vertical filtration membrane ultrafiltration tube is placed in a refrigerated centrifuge for first ultrafiltration, and the ultrafiltration conditions are as follows: 5000rpm, 4℃, and 15min. A first component with a molecular weight of ≥30kDa and a second component with a molecular weight of <30kDa are obtained. The second component is transferred into a 10kDa double vertical filtration membrane ultrafiltration tube using a 1ml pipette, and centrifuged again at 5000rpm and 4℃ for 15min. A third component with a molecular weight of <10kDa and a fourth component with a molecular weight of >10kDa and <30kDa are obtained. The fourth component is transferred into a new 2ml centrifuge tube using a 1ml pipette to obtain the blister beetle extract.

[0054] The prior art is based on fresh blister beetles as the extraction principle, while the present application first proposes a preparation method of a blister beetle extract using dry blister beetles as raw materials to effectively extract anticancer components in the dry blister beetles, thereby providing a new approach for extracting the blister beetle extract.

[0055] Example 2: Detection of the blister beetle extract

[0056] The blister beetle extract prepared by the preparation method described in Example 1 is used as the sample to be detected. The protein concentration of the sample to be detected is detected by referring to the method described in the instruction manual of the Bradford protein concentration determination kit (product number: P0006) of Bi Yun Tian, and the steps are as follows:

[0057] To the sample (1.5ml) in the centrifuge tube, 0.375ml of 5x protein loading buffer was added, shaken and mixed, and placed in a 100℃ metal bath for 15min. After the metal bath, the centrifuge tube was centrifuged at 8000rpm for 3min at 4℃ to obtain the protein sample. The protein sample and marker (Bi Yun Tian, P0060M) were added to the loading well of the Bradford protein concentration determination kit by using a pipette, and the total protein concentration in the sample was determined to be 1.52mg / ml.

[0058] First, the constant voltage of the electrophoresis apparatus was set to 80V for 30min, and then changed to 120V when the indicator reached the boundary between the concentrated gel and the separation gel. The electrophoresis was stopped when the indicator was 0.5cm from the bottom. The gel was removed from the gel plate and placed in a glass dish, and the electrophoresis buffer was washed off with pure water. 15ml of Coomassie brilliant blue staining solution was poured in, and the gel was stained on a shaker for 4h. The staining solution was discarded, and the residual staining solution was washed off with pure water. Enough elution solution was poured in, and the gel was eluted on a shaker. Three pieces of SDS-PAGE electrophoresis gel strip were cut, rinsed with pure water for 3 times, and placed in a low-temperature transport enzyme-free 1.5ml centrifuge tube to be sent to Shanghai Huoer Biotechnology Co., Ltd. for LC-MS / MS detection. The results are shown in Figure 1

[0059] As can be seen from Figure 1 , a clear protein band between 10-18kDa can be seen.

[0060] Example 3 Cell proliferation inhibition detection experiment of deilurus testaceus extract

[0061] SUM-159 cells, SK-BR-3 cells and MCF-7 / ADR cells were respectively placed in RPMI Medium 1640 culture medium containing 10%(V / V) FBS and 1% penicillin-streptomycin mixture, and cultured in a 37℃, 5% CO2 incubator to the logarithmic growth phase.

[0062] MCF-7 cells were cultured in MEM medium containing 10%(V / V) FBS and 1% penicillin-streptomycin mixture, and cultured at 37℃ to the logarithmic growth phase.

[0063] MCF-10A cells were cultured in MCF-10A cell-specific medium at 37℃ to the logarithmic growth phase.

[0064] MDA-MB-453 cells were cultured in Leibovitz's L-15 medium containing 10%(V / V) FBS and 1% penicillin-streptomycin mixture in a 37℃, 100% air-phase incubator to the logarithmic growth phase.

[0065] ​This embodiment is provided with 6 groups, which are: control (Control) group (replacing the extract of Pedilanthus tithymaloides with the same volume of PBS), 3 μg / mL extract of Pedilanthus tithymaloides group, 6 μg / mL extract of Pedilanthus tithymaloides group, 9 μg / mL extract of Pedilanthus tithymaloides group, 12 μg / mL extract of Pedilanthus tithymaloides group, and a blank group without cells. The extract of Pedilanthus tithymaloides groups of 3, 6, 12 and 18 μg / mL are prepared to the corresponding concentrations using complete culture medium.

[0066] Different cells in the logarithmic growth phase were taken, digested, counted, and then inoculated into plates and cultured for 24 h. When the cells adhered and reached 80%, the corresponding culture medium or drug was added, and the culture was continued for 24 h.

[0067] After the culture ended, the culture medium was discarded, 10% (V / V) MTT-containing medium was added to each well, and it was incubated in the incubator for 4 h. Then, the optical density (OD) was measured at 570 nm wavelength using an enzyme marker, and the survival rate was calculated, as shown in Figure 2

[0068] The calculation formula is: survival rate = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0069] As can be seen from Figure 2 Compared with the control group, the extract of Pedilanthus tithymaloides prepared in the application has a significant inhibitory effect on the cell viability of different breast cancer cells when the concentration is 12-18 μg / mL, indicating that the extract can inhibit the in vitro proliferation of breast cancer cells.

[0070] Example 4: Ferroptosis inhibitor rescue experiment

[0071] This embodiment is provided with five groups, which are: control (Control) group (adding the same volume of PBS and DMSO corresponding to one thousandth of the volume of the extract of Pedilanthus tithymaloides), 9 μg / mL extract of Pedilanthus tithymaloides group (CHP), 9 μg / mL extract of Pedilanthus tithymaloides + 1 μM DFO combined action group (CHP+DFO), 9 μg / mL extract of Pedilanthus tithymaloides + 1 μM Fer-1 combined action group (CHP+Fer-1), and a blank group without cells.

[0072] ​MCF-7 cells cultured to the logarithmic growth phase as described in Example 3 were digested and seeded in 96-well plates for 24 hours. Two combined treatment groups were pretreated with a cell death inhibitor (DFO or Fer-1) for 1 hour, followed by the addition of *Corydalis yanhusuo* extract. Other groups received their corresponding extracts or culture media simultaneously. After culturing for 48 hours, 100 μL of medium containing 10% (v / v) CCK-8 was added to each well, and the cells were incubated at 37°C for 2 hours. The optical density (OD) was measured at 450 nm using a microplate reader, and the survival rate was calculated using the formula: Survival rate = (Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%. The survival rate results were statistically analyzed. Figure 3 As shown.

[0073] Depend on Figure 3 It is evident that the ferroptosis-induced MCF-7 breast cancer cells induced by this extract can be rescued by the ferroptosis inhibitors DFO and Fer-1, indicating that this extract can induce ferroptosis in breast cancer cells.

[0074] Example 5: Ferric death-related indicators ROS and Fe 2+ Content detection

[0075] This embodiment sets up four groups: a control group (with an equal volume of PBS added), a 3 μg / mL stink bug extract group, a 6 μg / mL stink bug extract group, and a 12 μg / mL stink bug extract group.

[0076] (1) SUM-159 cells, MDA-MB-453 cells, MCF-7 cells, and mcf-7 / adr cells cultured to the logarithmic growth phase as described in Example 3 were digested, seeded in 96-well plates, and cultured for 24 h. After the cells adhered and reached 80%, the drugs were added as described in the above four groups, and the cells were cultured for another 24 h after drug addition.

[0077] After culture, the culture medium was discarded, and the DCFH-DA (reactive oxygen species fluorescent probe) reagent was diluted with serum-free culture medium at a volume ratio of 1:1000 to obtain a DCFH-DA dilution solution with a concentration of 10 μM. 100 μL of the DCFH-DA dilution solution was added to each well of a 96-well plate, and the plate was incubated at 37°C for 20 min, shaking every 5 min.

[0078] After the end of the culture, the culture in the 96-well plate was washed with serum-free cell culture fluid for 3 times, then 100 μL of 10 μg / mL Hoechst 33258 dyeing solution was added to each well for cell nucleus staining, the 96-well plate with the dyeing solution was placed in 37°C incubation for 20 min, the dyeing solution was discarded, and PBS was used for washing for 3 times. After the end of the washing, the 96-well plate was placed under a fluorescence microscope for taking pictures, and the results are shown in Figure 4 .

[0079] As can be seen from Figure 4 , compared with the control group, the red fluorescence in the SUM-159, MDA-MB-453, MCF-7 and MCF-7 / ADR cells was enhanced with the increase of the concentration of the extract, indicating that the Fe 2+ content in the breast cancer cells was increased.

[0080] (2) The SUM-159 cells, MDA-MB-453 cells, MCF-7 cells and mcf-7 / adr cells cultured to the logarithmic growth phase as described in Example 3 were subjected to digestion treatment, and were inoculated in a 96-well plate for culture for 24 h. After the cells were attached and reached 80%, the drug addition treatment was carried out according to the operation of the above four groups. After the drug addition, the culture was continued for 24 h.

[0081] The preheated serum-free cell culture medium was used to prepare 5 μM of RhoNox-1 working solution.

[0082] 100 μL of RhoNox-1 working solution was added to each well of the 96-well plate, and then incubated at room temperature for 25 min. After the end of the incubation, the RhoNox-1 working solution was absorbed, and the basic culture medium was used for washing for 3 times, 5 min each time. After the cell nucleus staining, 100 μL of 10 μg / mL Hoechst 33258 dyeing solution was added to each well, and then incubated at 37°C for 20 min. The dyeing solution was discarded, and PBS was used for washing for 3 times. The inverted fluorescence microscope was used for observation and taking pictures, and the results are shown in Figure 5 .

[0083] As can be seen from Figure 5 , compared with the control group, the green fluorescence of the extract group was stronger, and the green fluorescence was increased with the increase of the concentration of the extract, and the ROS level in the breast cancer SUM-159, MDA-MB-453, MCF-7 and MCF-7 / ADR cells was increased.

[0084] As can be seen from the above, the extract of aspongopus provided by the application shows good active inhibition effect on different types of breast cancer cells. The application provides a new idea for treating breast cancer by non-surgical approach and provides a new approach for reducing chemotherapy resistance. The preparation method provided by the application has low requirements for raw materials and instruments. The technical scheme of the application can solve the problem of difficulty in obtaining raw materials of the extract of aspongopus due to insufficient production of aspongopus, and can also solve the problem of high production cost of the extract of aspongopus caused by low utilization rate of aspongopus.

[0085] Although the above embodiment has described the application in detail, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained under the premise of no creativity, which all belong to the protection scope of the application.

Claims

1. A method for preparing an extract of Blatta lateralis, characterized in that, The method comprises the following steps: mixing the aspongopus raw material with water and sealing and refrigerating to obtain a first mixture; boiling the first mixture and separating the solid and liquid after cooling to obtain an aspongopus water extract; centrifuging the aspongopus water extract for the first time to obtain a first supernatant and a first precipitate, and collecting the first supernatant; heating the first supernatant to obtain a heated solution; centrifuging the heated solution for the second time to obtain a second supernatant and a second precipitate; mixing the second supernatant with a protease inhibitor to obtain a second mixture; ultrafiltrating the second mixture to collect components with molecular weight >10 kDa and <30 kDa to obtain an aspongopus extract.

2. The production method according to claim 1, wherein The aspongopus raw material comprises one or more of aspongopus dry insects and aspongopus residues; The aspongopus residues comprise insect residues after extracting aspongopus blood lymph; The mass-volume ratio of the aspongopus raw material to water is 40-60 g:100 mL.

3. The production method according to claim 1, wherein The refrigeration temperature is 4-6 ℃, and the refrigeration time is 20-28 h.

4. The production method according to claim 1, wherein The boiling time is 0.5-1.5 h.

5. The production method according to claim 1, wherein The rotation speeds of the first centrifugation and the second centrifugation are 10,000-14,000 rpm, respectively; the temperatures of the first centrifugation and the second centrifugation are 3-5 ℃, respectively; and the centrifugation times of the first centrifugation and the second centrifugation are 10-20 min, respectively.

6. The production method according to claim 1, wherein The heating treatment comprises metal bath treatment, alcohol lamp heating treatment or water bath treatment; When the heating treatment is metal bath treatment, the metal bath treatment temperature is 80-120 ℃, and the metal bath treatment time is 25-35 min.

7. The production method according to claim 1, wherein The volume ratio of the protease inhibitor to the second supernatant is 1-5:100-500.

8. The production method according to claim 1, wherein The ultrafiltration treatment comprises: first ultrafiltrating the second mixture by using a 30 kDa ultrafiltration device to obtain a first component with molecular weight ≥30 kDa and a second component with molecular weight <30 kDa; second ultrafiltrating the second component with molecular weight <30 kDa by using a 10 kDa ultrafiltration device to obtain a third component with molecular weight <10 kDa and a fourth component with molecular weight >10 kDa and <30 kDa, and collecting the fourth component to obtain the aspongopus extract.

9. The production method according to claim 8, wherein The first ultrafiltration treatment time is 20 min, the first ultrafiltration treatment rotation speed is 35,000 rpm, the first ultrafiltration treatment temperature is 4 ℃, and the 30 kDa ultrafiltration device comprises a 30 kDa double-vertical filtration membrane ultrafiltration tube; The second ultrafiltration treatment time is 20 min, the second ultrafiltration treatment rotation speed is 5,000 rpm, the second ultrafiltration treatment temperature is 4 ℃, and the 10 kDa ultrafiltration device comprises a 10 kDa double-vertical filtration membrane ultrafiltration tube.

10. Use of the aspongopus extract prepared by the preparation method of any one of claims 1-9 in the preparation of an antitumor drug.

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